Hexameric Storage Protein Research Articles

Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis

Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph.

Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes

As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

Gene expression in honey bee (Apis mellifera) larvae exposed to pesticides and Varroa mites (Varroa destructor)

Honey bee (Apis mellifera) larvae reared in vitro were exposed to one of nine pesticides and/or were challenged with the parasitic mite, Varroa destructor. Total RNA was extracted from individual larvae and first strand cDNAs were generated. Gene-expression changes in larvae were measured using quantitative PCR (qPCR) targeting transcripts for pathogens and genes involved in physiological processes, bee health, immunity, and/or xenobiotic detoxification. Transcript levels for Peptidoglycan Recognition Protein (PGRPSC), a pathogen recognition gene, increased in larvae exposed to Varroa mites (P<0.001) and were not changed in pesticide treated larvae. As expected, Varroa-parasitized brood had higher transcripts of Deformed Wing Virus than did control larvae (P<0.001). Varroa parasitism, arguably coupled with virus infection, resulted in significantly higher transcript abundances for the antimicrobial peptides abaecin, hymenoptaecin, and defensin1. Transcript levels for Prophenoloxidase-activating enzyme (PPOact), an immune end product, were elevated in larvae treated with myclobutanil and chlorothalonil (both are fungicides) (P<0.001). Transcript levels for Hexameric storage protein (Hsp70) were significantly upregulated in imidacloprid, fluvalinate, coumaphos, myclobutanil, and amitraz treated larvae. Definitive impacts of pesticides and Varroa parasitism on honey bee larval gene expression were demonstrated. Interactions between larval treatments and gene expression for the targeted genes are discussed.

Development of an RNA interference tool, characterization of its target, and an ecological test of caste differentiation in the eusocial wasp polistes.

Recent advancements in genomics provide new tools for evolutionary ecological research. The paper wasp genus Polistes is a model for social insect evolution and behavioral ecology. We developed RNA interference (RNAi)-mediated gene silencing to explore proposed connections between expression of hexameric storage proteins and worker vs. gyne (potential future foundress) castes in naturally-founded colonies of P. metricus. We extended four fragments of putative hexamerin-encoding P. metricus transcripts acquired from a previous study and fully sequenced a gene that encodes Hexamerin 2, one of two proposed hexameric storage proteins of P. metricus. MALDI-TOF/TOF, LC-MSMS, deglycosylation, and detection of phosphorylation assays showed that the two putative hexamerins diverge in peptide sequence and biochemistry. We targeted the hexamerin 2 gene in 5th (last)-instar larvae by feeding RNAi-inducing double-stranded hexamerin 2 RNA directly to larvae in naturally-founded colonies in the field. Larval development and adult traits were not significantly altered in hexamerin 2 knockdowns, but there were suggestive trends toward increased developmental time and less developed ovaries, which are gyne characteristics. By demonstrating how data acquisition from 454/Roche pyrosequencing can be combined with biochemical and proteomics assays and how RNAi can be deployed successfully in field experiments on Polistes, our results pave the way for functional genomic research that can contribute significantly to learning the interactions of environment, development, and the roles they play in paper wasp evolution and behavioral ecology.

Differential Gene Expression and Protein Abundance Evince Ontogenetic Bias toward Castes in a Primitively Eusocial Wasp

Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is “primitively eusocial” by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the foundation of the pathway divergence. Finally, the differential expression in Polistes larvae of genes and proteins also differentially expressed during queen vs. worker caste development in honey bees may indicate that regulatory mechanisms of caste outcomes share similarities between primitively eusocial and advanced eusocial Hymenoptera.

The role of storage proteins in colony-founding in termites

Hexameric storage proteins are widespread in insects and provide a source of amino acids when demand is high and intake is limited, such as during metamorphosis. We investigated the occurrence of storage protein levels in alates and in colony founding pairs in a dry-wood nesting species and two soil-nesting species of termites. Females and males contained similar quantities of storage protein at the time of the mating flight. Alates of soil-nesting species contained much higher levels of storage protein than the wood nesting species. After eggs hatched, protein stores plummeted in both sexes of both ecological types. Greater storage by alates of soil-nesting species is correlated with lack of access food during founding and the larger number of workers produced in their first broods.

A diapause pathway underlies the gyne phenotype inPolisteswasps, revealing an evolutionary route to caste-containing insect societies

Colonies of social wasps, ants, and bees are characterized by the production of two phenotypes of female offspring, workers that remain at their natal nest and nonworkers that are potential colony reproductives of the next generation. The phenotype difference includes morphology and is fixed during larval development in ants, honey bees, and some social wasps, all of which represent an advanced state of sociality. Paper wasps (Polistes) lack morphological castes and are thought to more closely resemble an ancestral state of sociality wherein the phenotype difference between workers and nonworkers is established only during adult life. We address an alternative hypothesis: a bias toward the potential reproductive (gyne) phenotype among Polistes female offspring occurs during larval development and is based on a facultatively expressed ancestral life history trait: diapause. We show that two signatures of diapause (extended maturation time and enhanced synthesis and sequestration of a hexameric storage protein) characterize the development of gyne offspring in Polistes metricus. Hexameric storage proteins are implicated in silencing juvenile hormone signaling, which is a prerequisite for diapause. Diverging hexamerin protein dynamics driven by changes in larval provisioning levels thereby provide one possible mechanism that can cause an adaptive shift in phenotype bias during the Polistes colony cycle. This ontogenetic basis for alternative female phenotypes in Polistes challenges the view that workers and gynes represent behavior options equally available to every female offspring, and it exemplifies how social insect castes can evolve from casteless lineages.

Differentially-expressed glycoproteins in Locusta migratoria hemolymph infected with Metarhizium anisopliae

Glycoproteins play important roles in insect physiology. Infection with pathogen always results in the differential expression of some glycoproteins, which may be involved in host–pathogen interactions. In this report, differentially-expressed glycoproteins from the hemolymph of locusts infected with Metarhizium anisopliae were analyzed by two-dimensional electrophoresis (2-DE) and PDQuest software. The results showed that 13 spots were differentially expressed, of which nine spots were upregulated and four were downregulated. Using MS/MS with de novo sequencing and NCBI database searches, three upregulated proteins were identified as locust transferrin, apolipoprotein precursor, and hexameric storage protein 3. These proteins have been reported to be involved in the insect innate immune response to microbial challenge. Due to the limited available genome information and protein sequences of locusts, the possible functions of the other 10 differentially-expressed spots remain unknown.

CDNA characterization and expression analysis of two arylphorin‐like hexameric protein genes from the diamondback moth, Plutella xylostella (L.)

We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investigated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin-like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the developmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome.

Structural basis for the presence of a monoglucosylated oligosaccharide in mature glycoproteins

Arylphorin is an insect hexameric storage protein. The structures of the oligosaccharides attached to this protein have recently been determined. However, their precise functions remain to be established. Proteolysis and MALDI MS studies disclose that the amino acid residues Asn196 and Asn344 are N-glycosylated with Glc 1Man 9GlcNAc 2 and Man 5–6GlcNAc 2 oligosaccharides, respectively. Interestingly, significant variations in the amounts of glycans involving Glc 1Man 9GlcNAc 2 are evident in arylphorins purified from larvae reared at different seasons. The data suggest that the metabolism of larvae and local protein structure contribute to glycan development. Three-dimensional model of the protein speculated that N-glycosidic linkage to Asn196 in the Glc 1Man 9GlcNAc 2 structure was buried inside the twofold axis of the hexamer, whereas oligosaccharide linkages to Asn344 were completely exposed to solvent. This finding is in agreement with previous biochemical data showing that limited Glc 1Man 9GlcNAc 2 was released by protein- N-glycosidase F under non-denaturing conditions, in contrast to Man 5–6GlcNAc 2 oligosaccharides.

CDNA cloning, expression, and characterization of an arylphorin-like hexameric storage protein, AgeHex2, from the mulberry longicorn beetle, Apriona germari.

An arylphorin-like hexameric storage protein, AgeHex2, cDNA was cloned from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae), larval cDNA library. The complete cDNA sequence of AgeHex2 is comprised of 2,088 bp encoding 696 amino acid residues. The AgeHex2 had four potential N-glycosylation sites. The AgeHex2 contained the highly conserved two larval storage protein signature motifs. The deduced protein sequence of AgeHex2 showed high homology with A. germari hexamerin1 (51% amino acid identity), Tenebrio molitor hexamerin2 (49% amino acid identity), T. molitor early-staged encapsulation inducing protein (43% amino acid identity), and Leptinotarsa decemlineata diapause protein1 (43% amino acid identity). Phylogenetic analysis further confirmed the AgeHex2 is more closely related to coleopteran hexamerins than to the other insect storage proteins. Northern blot analysis confirmed that the AgeHex2 showed fat body-specific expression. The cDNA encoding AgeHex2 was expressed as a 75-kDa protein in the baculovirus-infected insect cells. Furthermore, N-glycosylation of the recombinant AgeHex2 was revealed by tunicamycin to the recombinant virus-infected Sf9 cells, demonstrating that the AgeHex2 is N-glycosylated. Western blot analysis using the polyclonal antiserum against recombinant AgeHex2 indicated that the AgeHex2 corresponds to a 75-kDa storage protein present in the A. germari larval hemolymph.

Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper, Schistocerca americana

We identified a single hexameric storage protein in the grasshopper, Schistocerca americana, and monitored its abundance through the last larval instar and up until reproductive competence in adults of both sexes. This storage hexamerin, termed Schistocerca americana Persistent Storage Protein (saPSP) was the most abundant soluble protein in both larvae and adults. In both sexes, saPSP abundance started out low at the onset of the last larval instar and accumulated during feeding, peaking just prior to molting. Adults of both sexes contained significant amounts of saPSP after eclosion. In adult males, saPSP content dropped continuously after eclosion and was lowest once individuals reached reproductive maturity. In contrast, adult females depleted saPSP reserves during the first days of adulthood, but subsequently accumulated significant saPSP stores. In adult females, saPSP stores peaked just prior to the completion of egg provisioning. Given the overall patterns of abundance, saPSP has functions in both larvae and adults. In addition, the observed pattern of storage hexamerin accumulation differs from patterns of accumulation in the other known grasshoppers, Locusta migratoria and Romalea microptera, suggesting that significant functional diversity has evolved in storage hexamerins among the grasshoppers.

Molecular cloning, expression, and characterization of a cDNA encoding the arylphorin-like hexameric storage protein from the mulberry longicorn beetle, Apriona germari.

We describe here the cloning, expression, and characterization of a cDNA encoding the arylphorin-like hexameric storage protein from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The complete cDNA sequence of A. germari hexamerin (AgeHex) is comprised of 2,160 bp with 720 amino acid residues. The deduced protein sequence of AgeHex is most similar to Tenebrio molitor hexamerin2 (65.3%). Phylogenetic analysis further confirmed the AgeHex is more closely related to T. molitor hexmerin2 and T. molitor early-staged encapsulation inducing protein than to the other insect storage proteins. Southern blot analysis suggested the presence of A. germari hexamerin gene as a single copy and Northern blot analysis confirmed fat body-specific expression at the transcriptional level. The cDNA encoding AgeHex was expressed as a 80-kDa protein in the baculovirus-infected insect cells. Western blot analysis using the polyclonal antiserum against recombinant AgeHex indicated that the AgeHex corresponds to storage protein 2 (SP2) present in the A. germari larval hemolymph.

Female-specific expression of a hexamerin gene in larvae of an autogenous mosquito.

Fourth-instar larvae of the autogenous mosquito, Aedes atropalpus, synthesize three hexamerins or hexameric storage proteins which are distinguished by different methionine and aromatic amino-acid contents. One protein, Hexamerin-1.2 (AatHex-1.2) is only found in female larvae and pupae. In order to investigate the molecular basis for this sex-specific accumulation, we have cloned and sequenced the cDNA encoding AatHex-1.2 and isolated and sequenced over 1 kb of the 5' flanking region of the AatHex-1.2 gene. The AatHex-1.2 transcript encodes a 81.6-kDa hexamerin subunit which contains 19.8% phenylalanine, tyrosine and tryptophan and 8.6% methionine residues. The single-copy AatHex-1.2 gene consists of three exons and two small introns located at its 5' end. A 2.3-kb AatHex-1.2 mRNA accumulates only in female larvae and pupae and is expressed at very low levels in adult female mosquitoes. The temporal expression profile of this transcript is typical of other mosquito hexamerin genes, with rapid disappearance of the mRNA shortly after pupation. Hence this is the first observation of exclusively female-specific gene activity during preadult development of an insect. In the 5' flanking region of the AatHex-1.2 gene, we identified putative binding sites for transcription factors, such as GATA, C/EBP and Doublesex, typically involved in fat body- and female-specific gene activity in Diptera. These findings suggest that mechanisms for sex-specific transcription in the fat body may be well conserved between flies and mosquitoes.

Expression of larval hemolymph proteins ( Lhp) genes and protein synthesis in the fat body of greater wax moth ( Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18°C they enter diapause and cease further development for several months. During diapause a group of proteins (72–84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence ( Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90–100 days of diapause.

Hexameric storage proteins during metamorphosis and egg production in the diamondback moth, Plutella xylostella (Lepidoptera)

As in many Lepidoptera, Plutella xylostella adults do not feed on protein and females must use accumulated reserves to supply vitellogenin synthesis. Storage proteins were quantified in females and males from the late larval stage through day 4 of adult life. The level of storage protein peaked in the early pupal stage, with females having about twice as much as males. In males, the level fell through pupal development and dropped to a trace by one day after eclosion. In females, level of storage proteins fell until eclosion, and then rose dramatically within four hours after the molt to about 2/3 of the original peak level. This post-eclosion increase, which has not been reported previously in insects, suggests that adult females synthesize hexamerins to resequester amino acids. Subsequently, the level of storage proteins fell as vitellogenin appeared and eggs were laid. The ability to synthesize and sequester amino acids as storage proteins during the adult stage has wide-ranging implication for protein management in insects, particularly those that are long-lived and have flexible schedules of reproduction.

Expression profiles during honeybee caste determination.

Depending on their larval environment, female honeybees develop into either queens or workers. As in other polyphenisms, this developmental switch depends not on genomic differences between queens and workers but on the differential expression of entire suites of genes involved with larval fate. As such, this and other polyphenic systems can provide a novel tool for understanding how genomes and environmental conditions interact to produce different developmental trajectories. Here we use gene-expression profiles during honeybee caste determination to present the first genomic view of polyphenic development. Larvae raised as queens or workers differed greatly in their gene-expression patterns. Workers remained more faithful than queens to the expression profiles of younger, bipotential, larvae. Queens appeared to both downregulate many of the genes expressed by bipotential larvae and turn on a distinct set of caste-related genes. Queens overexpressed several metabolic enzymes, workers showed increased expression of a member of the cytochrome P450 family, hexameric storage proteins and dihydrodiol dehydrogenase, and young larvae overexpressed two putative heat-shock proteins (70 and 90 kDa), and several proteins related to RNA processing and translation. Large differences in gene expression between queens and workers indicate that social insect castes have faced strong directional selection pressures. Overexpression of metabolic enzymes by queen-destined larvae appears to reflect the enhanced growth rate of queens during late larval development. Many of the differently expressed genes we identified have been tied to metabolic rates and cellular responses to hormones, a result consistent with known physiological differences between queen and worker larvae.

TheDrosophila Lsp‐1βGene

In Drosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP-1), a mixed hexamer of three closely related subunits, Lsp-1 (alpha, beta and gamma) and larval serum protein 2 (LSP-2), a homohexamer of Lsp-2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of the Lsp-1 beta gene was determined for comparison with LSP-2 and a number of other arthropod hexamerins. The G + C content of the coding sequence is 55%, with 92.8% of the codons containing G or C in the third position. Conceptual translation of the Lsp-1 beta open reading frame revealed a 789-amino-acid polypeptide of 94465 Da. The amino acid sequence of Lsp-1 beta is 65.8% identical to that of calliphorin, the major hexamerin of the blowfly, Calliphora vicina, and only 35.2% identical to Drosophila Lsp-2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP-2 which is enriched to a lesser extent only in aromatic amino acids. Lsp-1 beta is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP-1 calliphorin and LSP-2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin.